PROJECT SUMMARY The overall objective of the ?Normal Aging Lung Cell Atlas? (NALCA), our response to RFA-HL-19-012 (Deciphering the Molecular Landscape of Lung Aging in Humans UO1), is to generate a compendium of the dynamic cell specific changes in mRNA, microRNA, and epigenetic marks that happen during lung aging with a focus on single cells and the alveolar microenvironment. We plan to use this compendium to generate a dynamic temporal regulatory model of normal human lung aging. The investigators on this application have significant expertise in lung genomics, epigenomics, bioengineering, aging, alveolar development, systems and computational biology. The premise of this proposal lies in the availability of resources, including normal human lungs at different ages, aged wild-type as well as aging relevant genetically modified mice, unique expertise in high throughput technologies including single cell RNAseq, laser capture microdissection, methylation profiling, proteomics and tissue engineering, and proven track record in developmental of analytical approaches that dissect temporal regulatory networks. It is also largely benefitting from synergisms with other non-overlapping NIH-NHLBI and NIH-NIA projects headed by the coinvestigators on this proposal. We will address the objectives of this project by the following specific aims: Specific Aim 1. To identify cell and microenvironment specific changes in coding and non-coding RNAs across human lung aging. We will perform single cell RNAseq of all the cells in the human lung across the spectrum of normal aging utilizing a unique collection of aging lungs. Analysis of lung microenvironments by LCM RNAseq will be performed in parallel on the same lungs, analyzing changes in alveoli, capillaries and inflammatory infiltrates. Aging methylation markers and cell specific validations will also be performed. Specific Aim 2. To identify key time points in lung aging using detailed temporal analysis of mouse lung aging. We will perform single cell RNAseq of lungs of wild-type mice of both sexes at multiple time points to identify the exact time points in which key changes in gene expression occur. We will then analyze these time points in detail, utilizing genetically modified mice with altered lifespan and aging mechanisms. Proteomic analysis will be performed on overlapping mouse and human key time points. Specific Aim 3. To develop a comprehensive transcriptional dynamic regulatory multicellular model of lung aging. We will use and extend our analytical tools to model specific dynamic signaling, regulatory networks, and cellular interactions, at the single cell level in lung aging. The model will provide insights and will be shared with the scientific community, through a highly interactive and intuitive web interface called Aging Lung Dynamic Regulatory Multicellular Model (AgeDREMM) that will allow visualization and sharing of our results.